Enhancing tomato plant immune responses to Fusarium wilt disease by red seaweed Jania sp.

The novelty of this study lies in demonstrating a new approach to control wilt diseases using Jania ethyl acetate extract. In the current investigation, the potential impacts of Jania sp. ethyl acetate extract (JE) on Tomato Fusarium oxysporum wilt (FOW) have been studied. The in vitro antifungal potential of JE against F. oxysporum (FO) was examined. GC–MS investigation of the JE revealed that, the compounds possessing fungicidal action were Phenol,2-methoxy-4-(2-propenyl)-,acetate, Eugenol, Caryophyllene oxide, Isoespintanol, Cadinene, Caryophylla-4(12),8(13)-dien-5à-ol and Copaen. Jania sp. ethyl acetate extract exhibited strong antifungal potential against FO, achieving a 20 mmzone of inhibition. In the experiment, two different methods were applied: soil irrigation (SI) and foliar application (FS) of JE. The results showed that both treatments reduced disease index present DIP by 20.83% and 33.33% respectively. The findings indicated that during FOW, proline, phenolics, and the antioxidant enzymes activity increased, while growth and photosynthetic pigments decreased. The morphological features, photosynthetic pigments, total phenol content, and antioxidant enzyme activity of infected plants improved when JE was applied through soil or foliar methods. It is interesting to note that the application of JE had a substantially less negative effect on the isozymes peroxidase and polyphenol oxidase in tomato plants, compared to FOW. These reactions differed depending on whether JE was applied foliarly or via the soil. Finally, the use of Jania sp. could be utilized commercially as an ecologically acceptable method to protect tomato plants against FOW.


Gas chromatography-mass spectroscopy (GC-MS) analysis
The metabolites present in the algal ethyl acetate extract was analyzed, counted, and identified using GC-MS, as explained by Sharaf et al. 14 .withminor modifications.Briefly, crude extract was dissolved in spectroscopy-grade methanol.GC-MS analysis was performed on Trace GC1310-ISQ mass spectrometer (Thermo Scientific, Austin, Texas, USA), using a direct capillary column (length 30 m, thickness 0.25 µm, internal diameter 25 mm).The oven temperature was started at 50 ℃ held for 5 min and ramped at 5 ℃ per min up to 230 ℃ and held for 2 min; 1 μL of the sample was injected at 250 ℃ using helium as a carrier gas, split at the ratio of 1:30.The mass spectrometer was run in the electron ionization (EI) mode at 200 ℃ and 70 eV with a scan range of 40 to 1000 m/z.The spectrum of the detected compounds was compared with the spectrum of the known compounds stored in the WILEY 09 (Wiley, New York, NY, USA) and NIST 11 library.The name, molecular weight, and chemical structure of the detected compounds were also determined.

Experimental location
The study was carried out at the experimental farm at Botany and Microbiology Department, Al-Azhar University, Cairo, Egypt.

Experimental scheme
Seedlings of the Solanum lycopersicum L. variety 023 were obtained from the Giza Agriculture Research Centre in Egypt at the age of four weeks.In a greenhouse, uniform seedlings were transplanted into plastic pots (40X40 cm) with a sand and clay mixture (1:3) weighing a total of 7 kg in the greenhouse.The pots were maintained at a constant 22/18 °C day/night and 70-85% relative humidity.JE was applied 7 days before inoculation with (FO).JE extract was given either through foliage or soil.The details of treatments (4 replicates for each treatment) include set I-healthy control (no fungus); set 2-FOW control; set 3-Healthy + JE (FS); set 4-FOW + JE (FS); set 5-Healthy + JE (SI) set 6-FOW + JE (SI); Plants were carefully uprooted 60 days after planting (DAP) and examined for the Disease index calculation and various characteristics mentioned below.

Vegetative development
Growth indicators such as root length (cm) and Stem height (cm) and number of leaves / plant were estimated post-harvest. www.nature.com/scientificreports/

Disease index (DI)
The disease symptoms were evaluated, and the disease index was calculated using a scale with five classes: 0 (no symptoms), 1 (slight yellowing of lower leaves), 2 (moderate yellowing of the plant), 3 (wilted plant), and 4 (severely stunted and destroyed plants).The disease index (DI) was determined using the formula: DI = [(1 × n1) + (2 × n2) + (3 × n3) + (4 × n4)] × 100 / (4 × Nt), where n1, n2, n3, and n4 represent the number of plants in each respective class, and Nt is the total number of plants evaluated.The severity of the FW disease and the percentage of protection provided by the JE were calculated using the following equation: Protection % = [(A-B) / A] × 100%.In this equation, A represents the Plant Disease Index in infected control tomato plants, and B represents the PDI in JE -treated tomato plants 6 .

Photosynthetic measurements
Fresh leaf tissue weighing 0.5 g was pulverised in 80% acetone using a pestle and mortar to determine the pigment content.After centrifuging for 5 min at 10,000 rpm (11180 g) (1118RCF) absorbance of filtrate was measured at 470, 652 and 665 nm to estimate chlorophyll 15 , and carotenoid 16

Measurement of phenolics
Total phenolics were determined by the process described 17 .The 1.5 mL Folin reagent solution received a 100 µL extract, and the mixture was then incubated at 25 ℃ for 1 min.1.5 mL of aqueous Na 2 CO 3 was added and allowed to sit at 25 ℃ for 90 min while kept in the dark.Absorbance was measured at 765 nm.

Assessment of proline
The Bates et al. 18 method was used to estimate proline.0.5 g of dry plant material were homogenized in 10 ml of 3% sulfosalicylic acid.The homogenate was filtered, and 2 ml of the filtrate was mixed with 2 ml of acid ninhydrin and 2 ml of glacial acetic acid.The mixture was heated for 1h, then immediately cooled in an ice bath.Four ml of toluene were added to the reaction mixture and mixed vigorously for 0.5 min.The toluene extract was then warmed to room temperature, and the absorbance was measured at 520 nm.

Test for antioxidant enzymes
The technique developed by Bergmeyer 19 was used to assess POD activity.Polyphenol oxidase (PPO) activity was recognized by a procedure of the Lavid et al. 20 .Two grams of fresh tomato plant were mixed with 10 ml of pH 6.8 phosphate buffer (0.1 M), homogenized, and preserved as an enzyme source.POD activity was measured by mixing 0.2 ml of the enzyme extract with 5.8 ml of 50 mM phosphate buffer (pH 7) and 2 ml of 20 mM H 2 O 2 .After adding 2 ml of 20 mM pyrogallol, the increase in absorbance was monitored at 470 nm.PPO activity was determined by mixing 125 µmol of phosphate buffer (pH 6.8), 100 µmol of pyrogallol, and 2 ml of the enzyme extract.After a 5 min incubation at 25 ℃, 1 ml of 5% H 2 SO 4 was added.The resulting color was measured at 430 nm.

Isozymes electrophoresis
Electrophoresis of isozymes was conducted using native polyacrylamide gel to observe modifications in response to treatments.The peroxidase (POD) and polyphenol oxidase (PPO) isozymes was evaluated using 4 .

Statistical analysis
The results presented are the means ± standard error of three replicates (n = 3).These results were statistically confirmed by analysis of variance (ANOVA LSD test at p < 0.05 was employed by CoStat to demonstrate the substantial variances between treatments.The findings (n = 3) were presented as an average for typical errors.

Plant collection
The plant collection and use were in accordance with all the relevant guidelines.

Algal identification
The red macroalga (Figs. 1 A-B) showing thallus and dichotomously branched, with segments and identified as Jania sp.

GC-MS analysis of the JE
The GC-MS analysis provides a typical spectral output for each compound found in the analysed samples.The GC-MS analysis of JE revealed that it contained approximately 35 compounds, with more than 70% are minor compounds.The highest peak area at 18.56 min with the presence of Eugenol (43.19%), followed by Phenol,2methoxy-4-(2-propenyl)-,acetate (35.89%),Caryophyllene oxide (5.77%), and Isoespintanol (3.69%), as shown in Table 1 and Fig. 2.

In-vitro antifungal activity of JE
The results observed in Fig. 3 indicate that JE has antifungal activity against FO, with an inhibition zone diameter of 20 mm.On the other hand, negative control had no effect on FO, which proves the exclusive activity of JE.Antifungal control (fluconazole) did not show activity against FO.

Photosynthetic pigments
Plant FOW resulted in a decline in components Chl. a and Chl.b.Comparing the samples to the healthy control plants, the carotene content did not significantly increase.As opposed to healthy control plants, a good recovery response was seen when healthy plants were treated with JE using various modalities.While JE through soil application recovered the loss of carotene content, JE through foliar spray was successful in recovering the loss of Chl. a and Chl.B (Table 4).

Total phenolics and proline
The total phenol and proline concentrations in the tomato plants were significantly higher with FOW compared to control plants (Fig. 5).The untreated healthy plants showed minimum values for total phenol content in comparison to FOW plants.Total phenol content, however, increased in JE -treated plants, reaching highest levels in infected plants treated with JE through foliar application, followed by JE through soil (Fig. 5).Infected plants treated with JE through soil treatment had the highest proline concentration, followed by plants treated with JE by foliar application (Fig. 5).

Activity of polyphenol oxidase (PPO) and peroxidase (POD)
In control plants, POD and PPO activity was barely detectable.Figure 6 clearly shows that POD and PPO activity increased dramatically in response to FO and/or JE.When JE was administered to FOW plants, the activity of POD and PPO also rose dramatically.The highest concentrations of these enzymes were discovered in infected plants that were treated with JE through foliar application initially, followed by JE through soil.

Antioxidant isozymes ( POD & PPO)
Native PAGE in (Fig. 7 ) and   The same six bands were recorded at the same Rf in infected plants treated with JE through different modes, such as soil and foliar spray, with two of them being moderated bands at Rf (0.634 and 0.903), while the other three bands were faint at Rf (0.470, 0.938, and 0.968), and one highly dense band at Rf values 0.845.Untreated control plants produced the fewest POD bands, 5, of which four were faint bands at Rf (0.634 and 0.903), three were faint at Rf (0.470, 0.634, 0.903, and 0.938), and one was moderately dense at Rf 0.845.
The PPO isozyme of tomato plant leaves at Rf contained four PPO isozymes (0.274, 0.492, 0.782, and 0.883).JE was applied to WOF plants for substantially overexpressed PPO, which recorded four bands, two of which were moderate bands at Rf (0.492 and 0.883), as well as two faint bands at Rf (0.274) and one extremely dense band at Rf (0.782).Healthy plants treated with JE through soil showed less expressed PPO that recorded 3 bands all of them was faint bands at Rf (0.492 0.782 and 0.883) as shown in Fig. 8 and Table 6.

Discussion
Numerous Notable bioactive ingredients have been discovered in red algae, including polysaccharides , lipids, glycosides, steroids, flavonoids, polyphenols, tannins, triterpenoids, alkaloids, and antheraquinones.They have a special ability to successfully halt the growth of many pathogenic microorganisms, particularly fungi 28 .
Protein, lipids, and carbohydrates are just a few of the main dietary components that constitute Jania.In aquaculture, the nutritional value of dietary seaweed supplements is determined by growth performance, feed consumption, and survival rate..Effective preventative measures have been created to lessen the harmful effects by eliminating the pathogen or enhancing plant resistance 31 .The addition of novel mitigating chemicals can enhance the growth and productivity of plant under biotic stress 32 .FO attacks a plant's internal vascular system .Instead of altering the entire soil micro flora, it is preferable to use biotic inducers as a safety measure for people and the environment to safeguard the spot where this fungus enters the plant 33 .
When plants were treated with JE, the proportion of disease severity decreased, and their resistance to and development of systemic resistance in plants increased.The application of JE under FOW significantly dramatically increased SL, RL, and NL.In the current study, FO dramatically decreased plant growth.The cumulative effects of FO on critical metabolic and enzyme functioning lead to a reduction in morphological characteristics and stresses hampering the cell cycle progression 34 .
Using biotic inducers for improving growth has been proposed as promising management technique for crop improvement.However, it is quite uncommon to find reports on JE advantageous effects on plants.The use of JE in the suggested method significantly increased the levels of chlorophyll and enhanced plant development.It was discovered in this study that using JE helped to advance the process of creating chlorophyll pigments.By mediating ROS scavenging and assisting in redox maintenance, the increased synthesis of carotenoids caused by the administration of JE possibly aided in photosynthetic defence.The use of bio stimulators increased the synthesis of carotenoids and chlorophyll 35 .It was shown that FOW dramatically enhanced POD and PPO activity 36 .Others have found similar further enhanced antioxidant activity 37 .The application of JE significantly enhanced POD and PPO activity.Previously, it has been observed that biotic inducers improve the performance of plants such as tomatoes as antioxidants 35 .The protection of photosynthesis, enzyme activity, and membrane integrity is facilitated by up regulated antioxidant enzyme activity, which maintains plant performance 38 .Although foliar applied JE was more helpful, both types of JE treatment had substantial effects.H 2 O 2 is eliminated by peroxidases at membranes 39 .Application of an amino acid-rich weed extract significantly enhanced the antioxidant performance of beans, leading to increased phenol, flavonoid accumulation 40,41 .Through improved stabilisation of cellular structures and enzyme activity in important metabolic pathways, JE-mediated rise in the proline content might have aided in cellular growth and maintenance 42 .The accumulated proline could be distributed throughout the cytoplasm to balance osmotic pressure from the vacuoles and protect bio macromolecules 43,44 (Supplementary information).

Conclusion
This study establishes that Fusarium oxysporum wilt (FOW) causes oxidative damage, leading to decreased physiological performance and growth in tomato plants.However, immune responses improved and antioxidant enzymes were up regulated when Jania sp.ethyl acetate extract (JE) was applied to the plants affected by FOW through foliage or soil.Additionally, plants exposed to JE exhibited an increase in osmoprotectants (proline) and phenolic compounds, both of which act as scavengers to eliminate excess reactive oxygen species during FOW infection.The study supports the beneficial application of JE in protecting tomato plants against FOW, although further research is necessary to elucidate the specific mechanisms involved.Table 6.Isomers of polyphenol oxidase enzymes (+ / −) and their Retention factor (Rf) in response to Fusarium, foliar application of Jania sp.and their interactions on tomato plants.

Table 2
and Fig.4reveal that the application of JE, either through foliar or soil, significantly reduced FOW PDI compared with infected control.Conversely, According to the results, infected control plants had a 91.6% PDI.foliar shoot by JE was the most effective treatment, reducing PDI by 20.83% and caused high protection by 77.25%.In the following, the soil irrigation by Jania algal extract reduced PDI by 33.33% and caused high protection by 63.61%.

Table 1 .
Major compound detected in GC Mass of JE.

Table 2 .
Effect of JE on DI.

Table 3
shows that JE treatments significantly affected various growth indices, including shoot length (SL), root length (RL), and leaf number (LN).Control tomato plants with FOW showed a marked decrease in SL, lower RL, and fewer leaves.Moreover, these plants lost more LN (60.1%),SL (26.8%), and RL (51.7% compared to healthy control plants).Conversely, healthy plants treated with JE through different methods, like soil and foliar spray, exhibited potential improvement in a variety of growth indices such as SL, RL, and LN.On the other hand, diseased plants treated with JE through various modes, such as foliar spray and soil application, shown encouraging recovery.In comparison to two modes, it was discovered that applying JE through foliar spray and soil irrigation to infected plants successfully restored the losses in SL(35 and 22%), RL (49.1 and 26.83%), and LN (31.6 and 12.5%).
*SL Shoot length, RL Root length, LN Leaves numbers.

Table 3 .
Morphological markers of a JE -treated tomato plant.

Table 4 .
Photosynthetic pigments of tomato plant treated with JE.

Table 5
showed Six POD isozymes at Rf values 0.470, 0.634, 0.845, 0.903, 0.938 and 0.968.POD activity was much higher in infected plants compared to healthy control plants.FOW Infected plants exhibited 6 bands, including three week bands at Rf values 0.903, 0.938 and 0.968, A moderate band at Rf 0.634 and highly dense band at Rf 0.845.

Table 5 .
Isomers of peroxidase enzymes (+ / −) and their Retention factor (Rf) in response to Fusarium, foliar application of Jania sp. and their interactions on tomato plants.
RF Control

healthy Control infected Healthy + JE Foliar Infected + JE Foliar Healthy + JE soil Infected + JE soil
GC-MS analysis of JE revealed that Eugenol, phenol,2-methoxy -4-(2-propenyl) -,acetate, caryophyllene oxide, isoespintanol are compounds recognized for having antifungal activity.The usefulness of a novel supplement, JE, in minimizing the detrimental effects of FOW tomato plants was examined in the current study.Wilt disease is regarded as one of the most detrimental biotic factors affecting development and productivity Figure 8.Effect of FO and/or JE and their interactions on PPO isozyme.I-healthy control (no fungus); 2-FOW infected control; 3-Healthy + JE (FS); 4-FOW + JE (FS); 5-Healthy + JE (SI) , and 6-FOW + JE (SI).